1. Field of the Invention
The present disclosure relates to microscopy, specifically to an illuminating optical system that illuminates a sample using total internal reflection. The invention may have broader uses including illumination for lithographic applications.
2. Description of Related Art
Fluorescent microscopy is a key tool in nearly all basic science labs and core facilities. Since the advent of genetically encoded tags, such as green fluorescent protein (GFP) and it's rainbow of color variants there has been an explosion in the use of fluorescent live cell imaging to major basic cellular processes. The easiest type of illumination is epi-fluorescent microscopy which typically uses a mercury arc lamp for Koehler illumination. Although this is the most popular fluorescent illumination method in terms of easy of use, it has the disadvantage that the whole cell is illuminated and the axial resolution is poor. To address the need for improved axial resolution and ‘optical sectioning’ the commercial confocal microscope was developed; common confocals are laser scanning or disk scanning. These instruments have an axial resolution of approximately 500 nm and are in wide use in the biological and material sciences. Its limitations are the requirement for lasers and specialized hardware and software and relatively poor sensitivity. Another form of light microscopy that provides thinner optical sectioning and much greater sensitivity is Total Internal Reflection Fluorescent Microscopy (TIRF; also called evanescent wave microscopy), as discussed below.
TIRF employs a totally internally reflected light (usually from a laser) to selectively illuminate and excite fluorophores in a very thin region (approximately 20-200+ nm) immediately adjacent to a glass-water (or glass-cell) interface. The evanescent field which ‘tunnels’ into the higher refractive index medium (e.g. aqueous or cell cytosol) exponentially drops in intensity along the z-axis and fluorophores away from the surface are not excited; the penetration depth depends on the wavelength, refractive indexes of the two mediums and the angle of the incident light. This optical method yields high signal-to-noise (S/N) images that is unmatched by any other light microscopy technique. It has greatly facilitated visualization of numerous cell surface process, such as signaling, exocytosis, endocytosis, migration, cytoskeleton, etc. The S/N is so high that single molecules can be directly studied, allowing biochemistry studies at the single molecule level in vivo and in vitro.
Penetration depth can be calculated by the below formulas:
Snell's law:n1sinθ1 = n2 sinθ2θ = angle of incidence, n = refractive indexCritical angle:At the critical angle, θc, θ2 = 90°; sin 90° = 1;n1sin θc = n2θc = sin-1 (n2/n1)             If      ⁢                          ⁢              n        1              =                  1.515        ⁢                                  ⁢        and        ⁢                                  ⁢                  n          2                    =      1.36        ,            θ      c        =                            sin                      -            1                          ⁡                  (                      1.36            1.515                    )                    =              63.85        ⁢        °             Evanescent field:            l      z        =                  l        0            ⁢              exp                              -            z                    /                      d            p                                          d      p        =          λ              4        ⁢                                  ⁢        π        ⁢                                                            n                1                2                            ⁢                              sin                2                            ⁢                              θ                1                                      -                          n              2              2                                           I = intensity, z = distance, λ = wavelength,dp = penetration depth
For TIR: n1>n2 (n=refractive index of medium1 and medium2; n1 is usually glass and n2 is usually aqueous). One can only use the formula for the evanescent field (3rd box), or to be more complete all 3 boxes can be used, wherein for TIR to occur the critical angle (defined above) must be exceeded.
With this technique, specimens (usually cells or tissue) are generally plated on glass coverslips and illuminated from the glass side with light that is incident at an angle greater than the so called ‘critical angle’ for transmission of light through the glass/cell cytosol interface. A prerequisite for TIRF is that the refractive index of the second medium (e.g. the cell cytosol (R.I. approximately 1.36-1.38) or water (R.I. approximately 1.33)) is less than the refractive index of the coverslip; the latter is typically 1.51 for standard coverslip glass, but other higher refractive index material such as sapphire may (R.I. approximately 1.78) be employed such as sapphire. Under these conditions a very shallow evanescent light field is generated that penetrates from the interface into the specimen. This is often termed “evanescent illumination”. The depth penetration of the illumination beyond the glass depends strongly on the incidence angle of the illumination light with respect to the glass surface.
One general class of instrumentation for TIRF imaging is “objective-type TIRF”. In this arrangement a single microscope objective lens is used for both evanescent illumination and also for recording images of fluorescent features. This technique is simple and cheap compared to alternative methods and has the advantage of allowing easy access to the side of the imaged specimen that is facing away from the objective lens.
A basic requirement of this method is that the numerical aperture of the objective lens is higher than the refractive index of the aqueous medium where total internal reflection occurs (typically water or cell cytosol), thus practically the numerical aperture (N.A.)>1.38 to achieve TIRF in cells (and generally the higher the better). Only in the last decade have the major microscopy manufactures been able to produce high N.A. lens (N.A. 1.45-1.65) so as to easily achieve TIRF with cells using ‘objective-type’ illumination. With sufficiently high N.A. objective lenses evanescent illumination can be generated from light passing near its extreme angular acceptance limit (in the back focal plane). Specifically, this corresponds to light passing through an annular band near the outer edges of objective entrance pupil. Light passing inside this ‘critical’ diameter has a sub-critical incidence angle at the interface of glass and specimen and is transmitted through the interface as regular, deep illumination. Light passing outside this ‘critical’ diameter can generate an evanescent illumination field.
This method of objective-TIRF illumination was first described in the following article: Stout, Al, Exelrod, D., “Evanescent Field Excitation of Fluorescence by EPI-Illumination Microscopy”, Applied Optics, 28 (24): 5237-5242, Dec. 15, 1989. This article describes four optical systems for evanescent illumination. In all of these arrangements an annular aperture is used to create an annular illumination pattern. This mask is located outside a conventional inverted fluorescence microscope and the pattern is relayed to the back pupil of the objective with lenses. A laser or an arc-lamp is indicated as the light source. The annular mask has a fixed position and a fixed diameter.
It should be noted that in one of the optical systems presented a conical axicon lens is used to improve light collection from an arc lamp source. This is the first mention of an axicon with regard to a TIRF imaging system. The axicon has a fixed light incidence angle.
TIRF is over twenty (20) years old and like the first confocal microscopes all setups were custom built, usually using a prism to couple the light into the sample. Again, only recently have microscopy companies developed high refractive index objective lens (1.45-1.65 N.A.) that permit one to do “objective-type” TIRF with cells. The latter has the advantage over “prism-type TIRF” that full access to the sample is permitted and could readily be adapted with these new lens. Objective-type TIRF also is more effective in collecting near-field emitted light than prism-type setups which typically use a water-immersion objective and collect far-field light that is transmitted through the aqueous medium.
TIRF have been extremely popular with scientists as they offer unmatched high-resolution imaging of processes near the cell surface. Analogous to confocal microscopes (including 2-photon microscopes) manufacturers have launched a whole series of instruments for this market (which are largely objective-type). Currently most objective-type TIRF instruments share a common design whereby laser light is focused onto a spot on the outer periphery of the back focal plane of the high NA objective lens. Although effective, major disadvantages have resulted from this approach: (1) the laser light easily generates interference effects, (2) the illumination is non-uniform and varies in penetration depth across the sample, (3) alignment of the system by the user is tricky as the spot can be placed in multiple equivalent positions, and (4) at deeper penetrations there is often a pronounced sideways scattering by the sample (due to illumination from the side and refractive index mismatch) which yields images that are smeared in a ‘coma’-type manner.
An alternative approach involves using a ring of light to illuminate the back focal plane. This has been achieved using a mask with an annular aperture to block the central portion of a Gaussian beam. While simple to implement the majority of the light is lost, requiring the use of a powerful laser. Second, the use of a wide enough annulus (e.g., 200 microns) to collect adequate light makes it hard to create a thin evanescent field, and thus the penetration depth is deeper than desired. Third, in order to change the angle of illumination requires insertion of a new mask with a different diameter annulus and recalibration. In application, this approach is neither efficient nor practical.
Another option would be to scan the back focal plane of a disk in a radial manner. This can remove in-homogeneities and decrease scattering, but either requires a spinning wedge or scanning of the back focal plane using galvomirrors or equivalent devices.
U.S. Pat. No. 6,992,820 describes a method of coupling the output of a laser, using an optical fiber, through a microscope, to the back pupil plane of an objective for the purpose of TIRF microscopy. The position of the relayed image of the optical fiber in the back pupil plane can be adjusted by moving the fiber tip. With this adjustment a range of incidence angles can be realized.
U.S. Pat. No. 7,042,638 is only slightly different than U.S. Pat. No. 6,992,820. The primary differences lie in the route by which light is routed into the microscope. Light from a fiber optic is routed to a single point in the back pupil plane of an objective.
Axicon optics were first introduced in the following paper. McLeod, J H, “The Axicon—A New Type of Optical Element, Journal of Optical Society AM, 44 (8): 592-597, 1954. McLeod coins the term “axicon” in this paper. It is defined broadly as: “all axicons are figures of revolution. An axicon has the property that a point source on its axis of revolution is imaged to a range of points along its axis.” Although conical lens are axicons the term axicon does not explicitly refer to conical lenses.
European Patent EP1211561A2 describes an illumination system for lithography that uses two axicons placed near a focal point in a scanned beam. The axicon spacing is adjustable and is used to control the coherence of light illuminating the field of an objective lens. A stationary integrating rod placed at a image plane is used to homogenize the light to achieve uniform illumination.
U.S. Pat. No. 5,675,401 describes an illumination system for lithography that uses two axicons placed inside a zoom lens, at a location where the beam through the axicon could either be converging or diverging depending on the zoom setting of the lens. The axicon spacing is adjustable and is used to control the coherence of light illuminating the field of an objective lens.
The present disclosure overcomes the disadvantages in conventional fixed axicon illumination system by providing an illumination system which uniquely provides: (1) a routine adjustment for incidence angle that is easily automated; (2) a small circular mask resulting in an insignificant light loss compared to a much larger loss from the annular aperture presented; and (3) elimination of the effects of laser speckle and interference fringes.
The present disclosure overcomes the disadvantages with regard to U.S. Pat. No. 6,992,820 by providing an illumination system which illuminates from 360 degrees around the optical axis of the objective lens. This reduces undesirable shadowing artifacts that can be caused by features in specimens. In addition, the present disclosure uniquely creates a uniform illumination field and eliminates speckle by temporally varying the illumination field.
The present disclosure relates to a novel system of total internal reflection fluorescent optics that is superior to conventional systems. The present disclosure offers better image quality, improved optical efficiency, and a unique depth penetration adjustment.
In addition, the present disclosure exhibits the following advantages over conventions system:                Improved uniformity of illumination        Reduced interference        Reduced ‘coma’ effect        Reduced shadowing from objects blocking illumination        Easy control of penetration depth        Ease of use and alignment        Designed for use with all objectives        Cost-effective        
Because the optics of the present disclosure illuminates a continuous 360 degree ring in the pupil of a TIRF objective, the illumination is extremely uniform. The shadowing problem inherent to illumination systems that have sources on only one side of the field of view is greatly reduced. It also reduces interference fringe effects. This results in much more homogeneous illumination and allows for better quantitative measurements to be made from TIRE images.
Additionally, the present disclosure allows for the radius of the illumination to be rapidly adjusted by adjusting the distance of the axicon from the focal point. This directly adjusts the illumination angle and the penetration depth of the evanescent field created. With this control a user has the ability to easily probe to different depths.